Over the past several years a growing family of leukocyte chemoattractant/activating factors, termed chemokines, has been described (Oppenheim, J. J. et al., Annu. Rev. Immunol., 9:617-648 (1991); Schall and Bacon, Curr. Opin. Immunol., 6:865-873 (1994); Baggiolini, M., et al., Adv. Imunol., 55:97-179 (1994)). Members of this family are produced and secreted by many cell types in response to early inflammatory mediators such as IL-1xcex2 or TNFxcex1. The chemokine superfamily comprises two main branches: the xcex1-chemokines (or CXC chemokines) and the xcex2-chemokines (CC chemokines). The xcex1-chemokine branch includes proteins such as IL-8, neutrophil activating peptide-2 (NAP-2), melanoma growth stimulatory activity (MGSA/gro or GROxcex1), and ENA-78, each of which have attracting and activating effects predominantly on neutrophils. The members of the xcex2-chemokine branch affect other cell types such as monocytes, lymphocytes, basophils, and eosinophils (Oppenheim, J. J. et al., Annu. Rev. Immunol., 9:617-648 (1991); Baggiolini, M., et al., Adv. Imunol., 55:97-179 (1994); Miller and Krangel, Crit. Rev. Immunol., 12:17-46 (1992); Jose, P. J., et al., J. Exp. Med., 179:881-118 (1994); Ponath, P. D., et al., J. Clin. Invest., 97:604-612 (1996)), and include proteins such as monocyte chemotactic proteins 1-4 (MCP-1, MCP-2, MCP-3, and MCP-4), RANTES, and macrophage inflammatory proteins (MIP-1xcex1, MIP-1xcex2). Recently, a new class of membrane-bound chemokine having a CX3C motif has been identified (Bazan, J. F. et al., Nature, 385: 640-644 (1997)). Chemokines can mediate a range of pro-inflammatory effects on leukocytes, such as chemotaxis, degranulation, synthesis of lipid mediators, and integrin activation (Oppenheim, J. J. et al., Annu. Rev. Immunol., 9:617-648 (1991); Baggiolini, M., et al., Adv. Imunol., 55:97-179 (1994); Miller, M. D. and Krangel, M. S., Crit. Rev. Immunol., 12:17-46 (1992)). Lately, certain xcex2-chemokines have been shown to suppress HIV-1 infection of human T cell lines in vitro (Cocchi, F., et al., Science (Wash. DC), 270:1811-1815 (1995)).
Chemokines bind to 7 transmembrane spanning (7TMS) G-protein coupled receptors (Murphy, P. M., Annu. Rev. Immunol., 12:593-633 (1994)). The principal human CXC chemokine receptors characterized to date include: CXCR1 (IL-8 Receptor type A (IL-8 RA)), which binds IL-8; CXCR2 (IL-8 RB), which binds a number of CXC chemokines including IL-8 and GROxcex1 (Murphy, P. M. and Tiffany, H. L., Science (Wash. DC), 253:1280-3 (1991); Beckmann, M. P., et al., Biochem. Biophys. Res. Commun., 179:784-789 (1991); Holmes, W. E., et al., Science (Wash. DC), 253:1278-1280 (1991)); an IP-10/Mig receptor designated CXCR3 (Loetscher et al., J. Exp. Med. 184:963-969 (1996)); and CXCR4 (also referred to as xe2x80x9cLESTRxe2x80x9d or xe2x80x9cfusinxe2x80x9d), which binds SDF-1 (Nagasawa et al., Proc. Natl. Acad. Sci. USA 93:14726-14729 (1996)). The known receptors for the CC or xcex2 chemokines Include CCR1, which binds MIP-1xcex1 and RANTES (Neote, K., et al., Cell, 72:415-425 (1993); Gao, J. L., J. Exp. Med., 177:1421-1427 (1993)); CCR2, which binds MCP-1 and MCP-3 (Charo, I. F., et al., Proc. Natl. Acad. Sci. USA, 91:2752-2756 (1994); Myers, S. J., et al., J. Biol. Chem., 270:5786-5792 (1995)); CCR3, which binds chemokines including eotaxin, RANTES and MCP-3 (Ponath, P. D., et al., J. Exp. Med., 183:2437-2448 (1996)); CCR4, which has been found to signal in response to MCP-1, MTP-1xcex1, and RANTES (Power, C. A., et al., J. Biol. Chem., 270:19495-19500 (1995)); and CCR5, which has been shown to signal in response to MIP-1xcex1, MIP-1xcex2 and RANTES (Boring, L., et al., J. Biol. Chem., 271 (13):7551-7558 (1996); Raport, C. J., J. Biol. Chem., 271:17161-17166 (1996); and Samson, M. et al., Biochemistry, 35:3362-3367 (1996)).
The precise expression of many of the chemokine receptors is not yet known, because specific mAbs are not available. For T cells, PCR or Northern blotting indicates that the known receptors for CC chemokines are expressed on subsets of T cells. Delineating exactly which subsets is an area of intense study, because chemokine receptor expression may explain the localization or migration of various cell types, such as TH1 or TH2 T cells or tissue homing subsets. It may also determine which T cells are infected with different strains of HIV-1. Despite the development of over 130 CD-defined specificities on leukocytes by the 5th International Leukocyte Workshop in 1993 (Schlossman, S. F., et al., Leukocyte Typing V, Oxford University Press, 1995), none of these are specific for chemokine receptors, pointing to the difficulty in making antibodies to these cell surface receptors.
The present invention relates to an antibody (immunoglobulin) or functional portion thereof (e.g., antigen binding fragment) which binds to a mammalian chemokine receptor 5 protein (also referred to as CKR-5 or CCR5) or portion of the receptor (anti-CCR5). In one embodiment, the antibody of the present invention has specificity for human CCR5 or portion thereof, wherein the antibody blocks binding of a ligand (e.g., RANTES, MIP-1xcex1, MIP-1xcex2, human immunodeficiency virus (HIV)) to the receptor and inhibits function associated with binding of the ligand to the receptor (e.g., leukocyte trafficking). For example, as described herein, antibodies of the present invention having specificity for human CCR5 or a portion thereof, can block binding of a chemokine (e.g., RANTES, MIP-1xcex1, MIP-1xcex2) to the receptor and inhibit function associated with binding of the chemokine to the receptor. In one embodiment, the antibody is monoclonal antibody 5C7 or a monoclonal antibody (mAb) which can compete with 5C7 for binding to human CCR5 or portion of human CCR5. In another embodiment, the antibody is monoclonal antibody 2D7 or a mAb which can compete with 2D7 for binding to human CCR5 or portion of human CCR5.
The present invention further relates to a method of inhibiting the interaction of a cell (e.g., leukocytes, T cells such as CD8+ cells, CD4+ cells and CD45RO+ cells, monocytes and transfected cells) bearing mammalian (e.g., human, non-human primate or murine) CCR5 with a ligand thereof, comprising contacting the cell with an effective amount of an antibody or functional portion thereof which binds to a mammalian CCR5 or portion of CCR5.
Another embodiment of the invention relates to a method of inhibiting the interaction of a cell bearing mammalian chemokine receptor 5 protein with a chemokine, comprising contacting said cell with an effective amount of an antibody or functional portion thereof which binds to a mammalian chemokine receptor 5 protein or portion of said receptor. In one embodiment of the method, the antibody or functional portion thereof is any one or more of 2D7, an antigen binding fragment of 2D7 or an antibody having an epitopic specificity which is the same as or similar to that of 2D7. Furthermore, the invention relates to a method of inhibiting a function associated with binding of a chemokine to the chemokine 5 receptor protein, comprising administering an effective amount of an antibody or functional portion thereof which binds to a mammalian chemokine receptor 5 protein or portion of said receptor. In one aspect of the method, the antibody or functional portion thereof is any one or more of 2D7, an antigen binding fragment of 2D7 or an antibody having an epitopic specificity which is the same as or similar to that of 2D7.
Another aspect of the invention is a method of identifying expression of a mammalian CCR5 or portion of the receptor by a cell. According to the method, a composition comprising a cell or fraction thereof (e.g., a membrane fraction) is contacted with an antibody or functional portion thereof (e.g., 5C7 or 2D7) which binds to a mammalian CCR5 or portion of the receptor under conditions appropriate for binding of the antibody thereto, and the formation of a complex between said antibody and said protein or portion thereof is detected. Detection of the complex indicates the presence of the receptor on the cell. The present invention also relates to a kit for use in detecting the presence of CCR5 or a portion thereof in a biological sample, comprising an antibody or functional portion thereof which binds to a mammalian chemokine receptor 5 protein or portion of said receptor, and ancillary reagents suitable for detecting the presence of a complex between said antibody and said protein or portion thereof.
Also encompassed by the present invention are methods of identifying additional ligands or other substances which bind a mammalian CCR5 protein, including inhibitors and/or promoters of mammalian CCR5 function. For example, agents having the same or a similar binding specificity as that of an antibody of the present invention or functional portion thereof can be identified by a competition assay with said antibody or portion thereof. Thus, the present invention also encompasses methods of identifying ligands or other substances which bind the CCR5 receptor, including inhibitors (e.g., antagonists) or promoters (e.g., agonists) of receptor function. In one embodiment, suitable host cells which have been engineered to express a receptor protein or variant encoded by a nucleic acid introduced into said cells are used in an assay to identify and assess the efficacy of ligands, inhibitors or promoters of receptor function. Such cells are also useful in assessing the function of the expressed receptor protein or polypeptide.
Thus, the invention also relates to a method of detecting or identifying an agent which binds a mammalian chemokine receptor 5 protein or ligand binding variant thereof, comprising combining an agent to be tested, an antibody or antigen binding fragment of the present invention (e.g., 2D7, an antibody having an epitopic specificity which is the same as or similar to that of 2D7, and antigen binding fragments thereof) and a composition comprising a mammalian chemokine receptor 5 protein or a ligand binding variant thereof. The foregoing components can be combined under conditions suitable for binding of the antibody or antigen binding fragment to mammalian chemokine receptor 5 protein or a ligand binding variant thereof, and binding of the antibody or fragment to the mammalian chemokine receptor 5 protein or ligand binding variant is detected or measured, either directly or indirectly, according to methods described herein or other suitable methods. A decrease in the amount of complex formed relative to a suitable control (e.g., in the absence of the agent to be tested) is indicative that the agent binds said receptor or variant. The composition comprising a mammalian chemokine receptor 5 protein or a ligand binding variant thereof can be a membrane fraction of a cell bearing recombinant chemokine receptor 5 protein or ligand binding variant thereof. The antibody or fragment thereof can be labeled with a label such as a radioisotope, spin label, antigen label, enzyme label, fluorescent group and chemiluminescent group. These and similar assays can be used to detect agents, including ligands (e.g., chemokines or strains of HIV which interact with CCR5) or other substances, including inhibitors or promoters of receptor function, which can bind CCR5 and compete with the antibodies described herein for binding to the receptor.
According to the present invention, ligands, inhibitors or promoters of receptor function can be identified in a suitable assay, and further assessed for therapeutic effect. Inhibitors of receptor function can be used to inhibit (reduce or prevent) receptor activity, and ligands and/or promoters can be used to induce (trigger or enhance) normal receptor function where indicated. Thus, the present invention also provides a method of treating HIV or inflammatory diseases, including autoimmune disease and graft rejection, comprising administering an inhibitor of receptor function to an individual (e.g., a mammal). The present invention further provides a method of stimulating receptor function by administering a novel ligand or promoter to an individual, providing a new approach to selective stimulation of leukocyte function, which is useful, for example, in the treatment of infectious diseases and cancer.
The present invention also relates to a method of detecting the susceptibility of a mammal to HIV. According to the method, a sample to be tested is contacted with an antibody or functional portion thereof which binds to a mammalian CCR5 or portion thereof, under conditions appropriate for binding of said antibody thereto, wherein the sample comprises cells which express CCR5 in normal individuals. Binding of antibody is detected using a suitable assay, and the binding of the antibody is indicative of the level of receptor expressed by the cells, which correlates with the susceptibility of the mammal to HIV. Thus, the method can be used to determine the expression level of CCR5 on the T cells of a susceptible but uninfected individual to determine the degree of risk to such an individual upon exposure to HIV. In another embodiment, a sample comprising a mammalian CCR5 protein, such as a cellular fraction or liposomes comprising said protein, can be used.
The present invention also encompasses a method of determining the prognosis for HIV in a mammal. According to the method, a sample to be tested is contacted with an antibody or functional portion thereof which binds to a mammalian CCR5 or portion thereof, under conditions appropriate for binding of said antibody thereto, wherein the sample comprises cells which express CCR5 in normal individuals. Binding of antibody is detected, and binding of antibody is indicative of the level of receptor expressed by the cells, which correlates with the prognosis for HIV in the mammal. In another embodiment, a sample comprising a mammalian CCR5 protein, such as a cellular fraction or liposomes comprising said protein, can be used.
Another aspect of the invention relates to a method of inhibiting HIV infection of a cell which expresses a mammalian CCR5 or portion thereof (e.g., monocytes, macrophages, dendritic cells or T cells such as CD4+ cells, CD8+ cells), comprising contacting the cell with an effective amount of an antibody or functional portion thereof which binds to a mammalian CCR5 or portion of the receptor.
Also encompassed by the present invention is a method of inhibiting (e.g., treating) HIV in a patient, comprising administering to the patient an effective amount of an antibody or functional portion thereof which binds to a mammalian CCR5 or portion of said receptor.
Another aspect of the invention also relates to a method of preventing or inhibiting HIV infection in an individual, comprising administering to the individual an effective amount of an antibody or functional portion thereof which binds to CCR5. According to the method, preventing HIV infection includes treatment in order to prevent (reduce or eliminate) infection of new cells in an infected individual or in order to prevent infection in an individual who may be, may have been or has been exposed to HIV. For example, individuals such as an HIV infected individual, a fetus of an HIV infected female, or a health care worker can be,treated according to the method of the present invention.
The present invention also encompasses a method of inhibiting leukocyte trafficking in a patient, comprising administering to the patient an effective amount of an antibody or functional portion thereof which binds to a mammalian CCR5 or portion of said receptor.